Cloning by nuclear transfer is performed by fusing a nuclear donor cell with an enucleated oocyte, typically a metaphase II oocyte. The resulting nuclear transfer embryo uses the donor cell DNA as the template for subsequent gene expression, thus producing a genetically identical clone of the donor cell line. Nuclear transfer research has increased our understanding of basic aspects of oocyte physiology and cell biology, and has tremendous potential for practical application in production animal agriculture and for the preservation of endangered species.
Nuclear transfer, like in-vitro fertilization, requires activation of an oocyte. In in-vitro fertilization, the oocyte is activated by entry of sperm into the oocyte. In nuclear transfer, activation is performed parthenogenetically, that is without the use of sperm. Most commonly, activation of the oocyte is obtained by introducing calcium ions into the activation medium, with or without other factors. In mice, exposure to calcium and magnesium ions has been reported to cause activation of oocytes. This procedure, however, has not proven to be effective in all species. For example, the procedure is ineffective in activating bovine oocytes. Susko-Parrish, U.S. Pat. No. 6,077,710. Susko-Parrish disclosed a method to obtain parthenogenetically activated bovine oocytes which included the steps of increasing divalent calcium cations in the oocyte and reducing phosphorylation of cellular proteins in the oocyte. The level of calcium ions in the oocyte is increased, according to the method of Susko-Parrish, by any method that increases intracellular cellular levels of divalent calcium cations, such as by electrical stimulation or addition of ionomycin to the activation medium. Reduction of phosphorylation of cellular proteins is obtained by addition of a serine-threonine kinase inhibitor, such as 6-dimethylaminopurine (DMAP) to the activation medium.
Nuclear transfer has been used successfully to clone offspring from sheep, cattle, goats, pigs, and mice. It is clear that different animals require different conditions in order to establish pregnancy by nuclear transfer. To date, nuclear transfer has been unsuccessful in establishing pregnancy in equine species.